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Abstract

Background

The chromodomain, helicase DNA-binding protein 5 (CHD5) is a potential tumor suppressor
gene located on chromosome 1p36, a region recurrently deleted in high risk neuroblastoma
(NB). Previous data have shown that CHD5 mRNA is present in normal neural tissues and in low risk NB, nevertheless, the distribution
of CHD5 protein has not been explored. The aim of this study was to investigate CHD5
protein expression as an immunohistochemical marker of outcome in NB. With this purpose,
CHD5 protein expression was analyzed in normal neural tissues and neuroblastic tumors
(NTs). CHD5 gene and protein expression was reexamined after induction chemotherapy in a subset
of high risk tumors to identify potential changes reflecting tumor response.

Results

We provide evidence that CHD5 is a neuron-specific protein, absent in glial cells,
with diverse expression amongst neuron types. Within NTs, CHD5 immunoreactivity was
found restricted to differentiating neuroblasts and ganglion-like cells, and absent
in undifferentiated neuroblasts and stromal Schwann cells. Correlation between protein
and mRNA levels was found, suggesting transcriptional regulation of CHD5. An immunohistochemical analysis of 90 primary NTs highlighted a strong association
of CHD5 expression with favorable prognostic variables (age at diagnosis <12 months,
low clinical stage, and favorable histology; P < 0.001 for all), overall survival
(OS) (P < 0.001) and event-free survival (EFS) (P < 0.001). Multivariate analysis
showed that CHD5 prognostic value is independent of other clinical and biologically
relevant parameters, and could therefore represent a marker of outcome in NB that
can be tested by conventional immunohistochemistry. The prognostic value of CHD5 was
confirmed in an independent, blinded set of 32 NB tumors (P < 0.001).

Reactivation of CHD5 expression after induction chemotherapy was observed mainly in those high risk tumors
with induced tumor cell differentiation features. Remarkably, these NB tumors showed
good clinical response and prolonged patient survival.

Conclusions

The neuron-specific protein CHD5 may represent a marker of outcome in NB that can
be tested by conventional immunohistochemistry. Re-establishment of CHD5 expression
induced by chemotherapy could be a surrogate marker of treatment response.

Introduction

Neuroblastic tumors (NTs) are embryonal cancers arising from neural crest derived
sympathetic nervous system precursors. These neoplasms are the most common extracranial
solid tumors in childhood and account for approximately 15% of all pediatric oncology
deaths [1].

Neuroblastoma (NB), the most undifferentiated form of NTs, embodies a heterogeneous
spectrum of diseases whereby patients with similar clinicopathological features exhibit
radically different outcomes ranging from spontaneous regression to inexorable progression.
Since treatment strategies vary from a "watchful waiting" approach to multimodal intensive
regimens, precise risk assessment is critical for therapeutic decisions. Various combinations
of prognostic markers have been used with success for risk group distinction, including
clinical, histologic and genetic factors, yet there remain cases where established
indicators of aggressiveness have demonstrated limited clinical utility. Additional
parameters are therefore needed for a more precise identification and therapeutic
targeting of high risk NB patients.

There is an apparent link between NB aggressiveness and specific genetic aberrations.
One of the most recurrent genetic alterations described is the deletion of the short
arm of chromosome 1 found in approximately 35% of NB [2]. The high incidence of chromosome 1p deletion in human cancer [2], with 1p36 deletion being the most common alteration [3], has led to an extensive search for 1p36 tumor suppressor genes. Recent findings
have identified the CHD5 gene as a candidate tumor suppressor [4,5] mapping to the smallest region of deletion (SRD) described in NB, 1p36.31 [6]. Evidence supporting CHD5 as a tumor suppressor is the recently reported strong promoter methylation and transcriptional
silencing of the remaining allele in 1p deleted NB cell lines [5]. Nevertheless, low or absent CHD5 expression levels have been found in NB cell lines lacking promoter methylation [7], 1p deletion, or inactivating mutations [6], suggesting other mechanisms by which CHD5 expression may be inhibited.

CHD5 is one of the nine members of the chromodomain helicase DNA-binding (CHD) family
of enzymes that belong to the ATP-dependent chromatin remodeling protein SNF2 superfamily
[8]. CHD protein structure is characterized by two N-terminal chromodomains and a SNF2-like
ATPase central domain that defines the chromodomain remodeling proteins [9,10]. The members of this evolutionarily conserved class of proteins play a critical role
in organizing the chromatin structure and accordingly, in chromatin based transcriptional
regulation of genes.

The aberrant expression of some of the CHD genes has been associated with human disease
processes like CHARGE syndrome, Hodgkin's lymphoma or dermatomyositis [8]. CHD5 mRNA expression, restricted to neuronal-derived tissues and the adrenal gland in normal
tissues [10], is basically absent in NB primary tumors with high risk features, MYCN amplification, advanced stage and 1p monosomy [5].

The distribution of CHD5 protein in NTs and normal neural tissues has not been explored.
Like neural tissue, NTs consist of two main cell populations, neuroblastic cells and
Schwann-like cells. The malignant potential of these tumors is inherently dependent
on the proportion of immature neuroblastic cells and the abundance of Schwann cell
stromal component, Schwannian stroma-poor undifferentiated NB being the most malignant.
CHD5 expression remains to be investigated in these two cell populations. In the present
study, we analyzed by immunohistochemistry normal neural derived tissues and NTs to
visualize CHD5 protein distribution within the different cell populations. Because
impaired CHD5 expression is associated with high risk NB tumors, we asked whether CHD5 protein expression
might serve as an immunohistochemical marker of outcome in NB. It is known that gene
expression pattern can change with treatment, for this reason, CHD5 gene and protein expression was re-examined after induction treatment in a set of
paired cases.

Material and Methods

Patients and tumor samples

A total of 90 primary tumor specimens (63 NB, 14 ganglioneuroblastomas (GNB) and 13
ganglioneuromas (GN)) (Additional file 1) were obtained at diagnosis from two institutions (Hospital Sant Joan de Déu (HSJD)
of Barcelona and Memorial Sloan-Kettering Cancer Center (MSKCC) of New York) together
with 12 high risk NB cases with available paired diagnostic and post-chemotherapy
tumor specimens. An independent set of 32 NB tumors was obtained from Children's Hospital
of Boston and Dana-Farber Cancer Institute (CHB/DFCI) for data validation analysis.
Non-tumor samples (fetal brain, adult cerebral cortex, adult cerebellum, adrenal gland,
bone marrow, spinal cord and sympathetic ganglion) were also included in this study.

NB risk assessment was defined by the International Neuroblastoma Staging System (INSS)
[11]. NB stages 1, 2, 3 (MYCN non-amplified) and 4s were uniformly treated without use of cytotoxic therapy, when
possible. Stage 4 and stage 3 MYCN amplified NB patients were treated according to N5, N6 or N7 protocols. This study
was approved by the Institutional Review Boards and informed consent was obtained
before collection of samples.

Tumors were assessed by a pathologist (M.S.), only tumors with >70% viable tumor cell
content were included in the study.

Slides were examined by a pathologist (M.S.) using an Olympus BX41 light microscopy
to assess staining and score both percentage of positive cells and staining intensity
(0, negative; 1, weak; 2, strong and 3, very intense staining). Integer values were
assigned to the proportion of positive cells (<25% = 1; 25-75 = 2; >75% = 3). Intensity
and positive cell values were multiplied to provide a single score for each case.

RNA isolation and cDNA synthesis

Total RNA was isolated from snap frozen samples and cell lines using Tri Reagent (Sigma,
US), following manufacturers' protocols. cDNA was synthesized from 1 μg total RNA
using random primers and M-MLV reverse transcriptase (Promega, US) as previously described
[12].

Quantitative Real-time Polymerase Chain Reaction (qRT-PCR)

Quantification of transcript levels, using the ΔΔCT relative quantification method, were performed on an ABI Prism 7000 Sequence Detection
System with TaqMan® Assay-on-Demand Gene Expression products (Applied Biosystems, US), as previously reported
[12].

Statistical analysis

Comparisons between immunohistochemical results were performed by means of the log-rank
test. qRT-PCR transcript levels were normalized by z-score transformation to enable
a correlation analysis with the immunostaining score values. Correspondence between
immunoreactivity and mRNA expression levels within the same samples was examined using
the Spearman's correlation coefficient analysis. Statistical analyses for qualitative
variables were performed by means of the Fisher's exact test and U Mann-Whitney test
for quantitative or ordinal variables. Overall survival (OS) and event-free survival
(EFS) probabilities were estimated using the Kaplan-Meier method. Multivariate Cox
regression models were used to examine the prognostic significance of CHD5, INSS stage,
age at diagnosis, MYCN status and 1p LOH. Each variable consisted of two groups: "INSS stage" consisted of:
(1) ST1, 2, 3 and 4s, and (2) ST4; "age" (at diagnosis): (1) ≤ 12 months (2) > 12
months; "MYCN": (1) MYCN non-amplified (2) MYCN amplified; "LOH": (1) no LOH (2) LOH. Predictive Positive and Negative Values (PPV
and NPV) were used for a descriptive comparison between CHD5 expression and MYCN and 1p LOH. All reported P-values are two-sided. P-values ≤0.05 were considered statistically
significant. Statistical analysis was performed with SPSS 15.0 package (SPSS, Chicago,
IL).

CHD5 expression was evaluated in brain cortex specimens and in NB cell lines by immunoblot
analysis. CHD5 protein (250-260 kDa) was detected only in brain cortex specimens,
both in the total protein extract and in the nuclear fraction. No CHD5 protein was
detected in the cytoplasmic fraction of all the analyzed specimens or in NB cell lines
(Figure 1B).

The described immunohistochemical assays were performed using two different batches
of the anti-CHD5 antibody (T00251-A1 and T00251-A02). Both batches performed consistently
across many repeats, further supporting the validity of our results (Additional file
2). The specificity of the anti-CHD5 antibody was further validated on mouse xenografts
of human NB cell lines (SK-N-JD, SK-N-LP and SK-N-AS). All the xenografts were found
to be negative for CHD5 staining (Additional file 2).

Additonal file 2.A. Immunohistochemical staining of FFPE sections of two immunopositive neuroblastic
tumors using two different batches of the anti-CHD5 antibody (T00251-A1 and T00251-A02);
B. Immunochemical assay with the anti-CHD5 antibody (Strategic Diagnostics, DE) on
mouse xenografts derived from human NB cell lines. The specificity of the anti-CHD5 antibody was validated by immunohistochemical assays
on FFPE sections of mouse xenografts of human NB cell lines (SK-N-JD, SK-N-LP and
SK-N-AS). In these NB cell lines CHD5 gene expression is very low or absent (data not shown), similar to previously reported
data (ref. 5, ref. 10). Two different anti-CHD5 antibody batches (T00251-A1 and T00251-A02,
Strategic Diagnostics, DE) were tested. Ganglioneuroblastoma FFPE tissue sections
were used as positive control samples. All the analyzed xenographs were composed nearly
exclusively (>95%) of neuroblastic cells exhibiting no CHD5 nuclear staining and faint
cytoplasmic staining (when present). Only few (<5%) immunopositve cells were observed
in the SK-N-LP xenograft. However, viable tumor cells in the SK-N-LP xenograft where
negative for CHD5 nuclear staining, similar to SK-N-JD and SK-N-AS. These results
were comparable to the immunostaining pattern observed in undifferentiated high risk
NB tumors. The GNB ganglionar cells showed intense nuclear and diffused cytoplasm
immunostaining.

Additional file 5.Cox multivariate análisis. Cox multivariate regression analysis has been performed using clinical and biological
variables currently used in risk stratification of NB patients (INSS stage, age at
diagnosis, MYCN status and 1p LOH) in combination with the CHD5 IHC. The analysis has been performed
sequentially, adding one variable at each step, in order to assess how the presence
of each variable influences the performance of CHD5. CHD5 IHC remained statistically
significantly associated with overall survival in all the analyses, except when the
1p LOH parameter is included in the overall survival analysis. This is due to the
strong association of the expression of CHD5, located on 1p36, with chromosome 1p status. All the rest of variables, except for
MYCN amplification, were not statistically significant. For event free survival analysis,
CHD5 IHC is the only variable that remained statistically significant along the whole
analysis, even in the presence of 1p LOH. IHC = Immunohistochemical analysis; INSS
= International Neuroblastoma Staging System; HR = hazard ratio; CI = confidence interval.
P-values are two sided.

Tumor histology and gene expression can change with treatment as a result of important
changes in cellular processes. We investigated the effects of induction chemotherapy
(3 cycles) on CHD5 expression in 12 high risk NB cases with available paired diagnostic and post-chemotherapy
tumor specimens for qRT-PCR and immunohistochemical analyses. At diagnosis all these
tumors (2 locoregional and 10 stage 4 NB) displayed low CHD5 mRNA expression and negative immunostaining. Following induction chemotherapy, a significant
increase of CHD5 transcript and CHD5 positive nuclear staining was detected in 6/12 specimens, together
with therapy-induced morphological changes (increased cytoplasm and ganglion-like
cell morphology) (Figure 4A and 4C; cases #1-6). All these patients achieved an initial complete or very good response
to cytotoxic therapy (chemo- and radiation therapy). At the time of analysis, 5/6
patients were alive with a mean follow-up of 35.62 months (Figure 4B). One case, stage 4 MYCN amplified, progressed after a good initial response to chemotherapy and died of refractory
bone marrow disease (Figure 4C; case #6). Bone marrow aspirate smears of this patient exhibited widespread tumor
dissemination with CHD5 negative neuroblast aggregates (data not shown).

These observations suggest a relationship between CHD5 expression reactivation and
response to induction therapy and subsequent patient outcome.

Discussion

Gene expression of CHD5, an ATP-dependent chromatin remodeling enzyme, has been reported
to be restricted essentially to the nervous system [8,10]. We describe for the first time that CHD5 is a neuron specific protein in normal
neural tissue, with variable immunostaining intensity and intracellular localization
among the neuron types of the cerebral cortex. Recent evidences suggest that the diverse
neuron cell classes derive from distinct embryonal germinal zones and are characterized
by specific cell signaling systems that regulate neural stem cells throughout the
developing brain [13-15]. Thus, neuronal cells adopt a brain layer fate determined by their molecular profiles
[14]. While we did not observe a layer specific distribution of CHD5 in the cerebral cortex,
we did note an association of CHD5 expression with neurons with distinct morphological,
physiological and neurochemical features.

In normal neural tissue, glial cells appeared consistently devoid of CHD5 expression. In human glial tumors, chromosome arm 1p allelic loss is a frequent genetic
abnormality, especially in oligodendrogliomas (70-85%) and astrocytomas (20-30%) [16]. Recently, low levels of CHD5 expression have been reported in gliomas with 1p deletion, whereas nondeleted tumors
displayed expression levels comparable to normal brain [4]. Thus, deletion of CHD5 has been proposed as an initiating event in gliomas [4]. Our findings, however, suggest that the role of CHD5 as a tumor suppressor in glial
tumors needs further investigation.

NTs are embryonal cancers that are assumed to originate from primitive sympathetic
neuroblast aggregates located in neural crest derived sympathetic nervous system.
We observed how primitive neuroblast aggregates found in fetal adrenal gland specimens
generally lack CHD5 expression. Interestingly, only a few cells were found with a
variable degree of nuclear reactivity in larger aggregates. To date, the fate of these
immature neuroblastic aggregates remains unsolved, and spontaneous involution and
cell maturation have been proposed [17]. The immunoreactivity observed in a small proportion of neuroblasts within these
islets could suggest the establishment of CHD5 expression prior to their disappearance;
however, no evident differentiating features were observed in these immunopositive
cells that suggested the activation of the maturation process.

In NTs, CHD5 is essentially expressed in the nucleus of differentiating neuroblastic
cells and ganglion cells, and absent in the Schwannian stromal component. However,
the most intense immunoreactivity was observed in stage 4s NB, a rare subgroup of
histologically undifferentiated, highly proliferative, metastatic tumors with a high
incidence of spontaneous regression, affecting young infants. Accurate distinction
of spontaneously regressing infant NB from high risk infant stage 4 can be difficult,
but critical for therapeutic decisions. In our hands, the intensely positive CHD5
nuclear staining enabled a clear distinction of stage 4s NB from stage 4 NB, which
was consistently immunonegative. These results are consistent with our previous gene
expression profiling study, where similar differential CHD5 expression profiles were observed amongst infants with disseminated NB subgroups [18]. Thus, CHD5 immunohistochemical staining may be clinically useful for a more accurate
characterization of disseminated infant NB.

In NB, CHD5 nuclear staining was strongly associated with established favorable prognostic
variables like low clinical stage, age at diagnosis <12 months and favorable histology.
Our findings suggest that CHD5 protein expression may accurately define NB risk groups
and may, therefore, be a prognostic marker. Evidence is provided by the statistically
significant association found between high CHD5 immunoreactivity and favorable OS
and EFS. These results are consistent with recent studies reporting a strong association
of CHD5 mRNA levels with patient outcome in NB [5,10]. Furthermore, Cox multivariate analyses suggest that the prognostic value of CHD5
protein expression is independent of other clinical and biological variables currently
used in risk stratification of NB patients and could therefore represent an immunohistochemical
marker of prognosis in NB.

Currently, risk stratification of NB patients is performed by combining different
markers with strong prognostic impact, including patients' age at diagnosis, tumor
stage, genomic amplification of the oncogene MYCN, copy number alterations of chromosomal regions 1p, 11q and 17q, tumor DNA content
[1,19] and Shimada histological score [20]. However, despite elaborate risk stratification strategies, outcome prediction in
neuroblastoma is still deficient. In recent years, to improve risk assessment additional
prognostic indicators such as gene-expression signatures [21-23], combined genomic and molecular signatures [24] or expression levels of single candidate genes, e.g., Trk (NTRK) family of neurotrophin receptors [25,26], FYN [27], PRAME [28] and ZNF423 [29], have been associated with NB clinical behavior. Expression of the Trk family receptors
has been the most extensively characterized marker in NB and has been found to be
consistently correlated with the biology and clinical behavior of NB. Based on our
results, there is an apparent similarity between the expression patterns of CHD5 and TRKA in NB and their patterns of association with NB disease outcome. TRKA expression has
been reported to be high in biologically favorable NB tumors and inversely associated
with MYCN amplification [30]. The prognostic value of the immunohistochemical detection of TrkA has also been
examined and reported to be high, especially in combination with Ha-Ras expression
pattern [31,32]. Further IHC studies have correlated the lack of TrkA expression with metastatic
malignant NB [33]. However, in the latter study, 34% of the patients with stage 4 NB displayed TrkA
expression, a subset of which died of aggressive metastatic disease despite TrkA expression
[33,34]. In our study, the majority of stage 4 NB either lacked CHD5 immunoreactivity (83%)
or exhibited weak nuclear staining (13%), a high risk phenotype according to our scoring
system. Only one stage 4 tumor was found to be clearly immunoreactive for CHD5; at
the time of analysis the patient is alive, 29 months from diagnosis. These observations
further confirm CHD5 as a powerful prognostic marker that could complement other known
markers such as age at diagnosis, stage, MYCN status, cellular DNA content, 1p deletion and tumor histology. However, the potential
clinical use of this marker must be tested in larger, prospective cohorts.

It is known that tumor histology and gene expression can change with treatment as
a result of important changes in cellular processes, e.g., induced tumor differentiation,
DNA repair, apoptosis and tissue necrosis. Undifferentiated NB occasionally exhibit
neuroblastic maturation in response to chemotherapy. Assessment of CHD5 gene and protein expression in NB post-therapy specimens revealed that tumors with
evident neuroblastic maturation showed both CHD5 gene and protein reactivation. Notably, none of these tumors harbored 1p deletion.
Conversely, in tumors where minimal or no morphological changes were observed in the
post-treatment specimens, low CHD5 expression persisted. These observations suggest the existence of a subset of tumors
within high risk NB where CHD5 expression can be reactivated from the silenced state by standard chemotherapy. Remarkably,
when post-therapy reactivation was observed, CHD5 expression was largely associated with disease response to cytotoxic induction therapy
and subsequently with longer patient OS. All 12 patients included in the study received
the same treatment, nevertheless some tumors failed to respond. At present, treatment
response in NB is routinely evaluated by monitoring urine levels of catecholamine
and its metabolites (VMA/HVA ratio) and by estimating the decrease in the size of
measurable lesions with conventional imaging modalities, such as computed tomography
(CT) or magnetic resonance imaging (MRI). At the time of second-look surgery, the
degree of induced tumor cell differentiation and the extent of necrosis can also be
useful to estimate treatment response. However, no biological markers for tumor chemotherapy
responsiveness have been reported in NB. The use of such biomarkers would make chemotherapy
more effective for individual patients by allowing timely changes of therapy in the
case of nonresponding tumors. Furthermore, markers reflecting tumor response can function
as surrogates of long-term outcome. Taking into account the small cohort of cases
that may have led to an overestimation of the data, our findings would suggest that
restoration of CHD5 expression could be a surrogate marker of treatment response that
can be clinically useful to identify patients that do not benefit from conventional
treatment. These results warrant further investigation in a larger cohort of uniformly
treated patients.

In summary, we report that the differential expression of the neuron-specific protein
CHD5 accurately defines NB risk groups and may represent a marker of outcome in neuroblastoma
that can be tested by conventional immunohistochemistry. In high risk NB patients,
re-establishment of CHD5 expression following chemotherapy should be tested prospectively
as a surrogate marker of treatment response.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

CL, IG and JM are responsible for the initial conception and overall hypothesis of
this study. IG, GM and CL are responsible for the design of this manuscript, including
the original draft and subsequent revisions. IG, GM, ER, MS, TG, JR, NKC, CdT, JM
MK, RG, AAP and CL were involved with the interpretation of data, draft and revision
of this manuscript. CdT provided guidance for many of the experiments. NKC, TG, MK,
RG and AAP are responsible for the procurement and cryopreservation of NBT tissue
specimens derived from MSKCC and CHB/DFCI. ER, IG, GM, JM and CL were responsible
for the procurement and cryopreservation of NBT tissue specimens derived from the
Spanish institutions. ER, IG GM, CL and MS are responsible of inmunohistochemical
analyses. MS evaluated tumour specimens for staging classification, tumour content.
JM and CL are responsible for patient clinico-biological database management. CC,
GM, PG and ER are responsible for the in vivo study. IG, GM and CL are responsible for the quantitative PCR experiments and inmunoblotting.
JR and CL are responsible of statistical analyses. All authors were involved in the
drafting and revisions for this manuscript. All authors read and approved the final
manuscript.